Purification and characterization of bovine tissue factor.
Tissue factor (tissue thromboplastin, factor III), an initiator of coagulation, has been purified 142,000-fold to homogeneity from bovine brain. The protein is an integral membrane glycoprotein with an apparent molecular weight of 43,000 as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The apoprotein was first purified by extraction with Triton X-100 and repeated preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Antiserum was produced against a few micrograms of purified apoprotein and was used to construct an immunoadsorbent column. The column was then used for affinity purification of the apoprotein directly from the Triton X-100 extract, thereby significantly increasing the amount of purified protein produced. The purification scheme may be generally useful for the rapid and large scale purification of membrane proteins. Tryptic digestion of the apoprotein in Triton X-100 cleaved a peptide of approximately 3000 daltons without affecting the activity. The activity was recovered directly from stained SDS polyacrylamide gels, and the profile of recovered activity corresponded directly with the stained bands. The activity shifted along with the protein band following tryptic digestion, thus demonstrating that the protein observed on the gels is tissue factor. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipid. Optimal activity was observed at phospholipid to protein ratios (w/w) greater than 450:1.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification; D001923:Brain Chemistry; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010743:Phospholipids / Q000302:isolation & purification; D013925:Thromboplastin / Q000302:isolation & purification*; D014357:Trypsin
D000596:Amino Acids; D001059:Apoproteins; D010446:Peptide Fragments; D010743:Phospholipids; D013925:Thromboplastin; D014357:Trypsin